Determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid by high-performance liquid chromatography,using on-column concentration and electrochemical detection

Chromatographic conditions for the determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid are described, involving simple pretreatment of the sample. Electrochemical detection was used. The linear range of the method is more than 10³. Recovery from plasma and spinal fluid is 100%, and the detection limit of the method is 2·10^?9M, sufficient for the detection of endogenous plasma and spinal fluid levels. The detection conditions are discussed. Endogenous concentrations of the compound in plasma and spinal fluid were determined and correlated with a folate bioassay. Plasma concentrations have been shown after the administration of leucovorin which is used in anticancer therapy.     

Detection and Pharmacokinetics of Alginate Oligosaccharides in Mouse Plasma and Urine after Oral Administration by a Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) Method

A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 μg/ml at 5 min and 425.5 μg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse.     

Determination of BAY x 7195, a novel leukotriene D4 antagonist,in human plasma by high-performance liquid chromatography with post-column photo derivatisation and fluorescence detection

Methods to determine plasma concentrations of the leukotriene D₄ antagonist BAY x 7195 by HPLC with post-column photo derivatisation and fluorescence detection are described. Following dilution and centrifugation plasma supernatant is injected onto the HPLC system allowing the selective determination of the drug with a limit of quantitation (LOQ) of 10 μg/l (method A). Sensitivity was further enhanced to a LOQ of 0.6 μg/l by employing solid-phase extraction whereby the analyte concentration in the injection solution was increased (method B). Data on recovery, accuracy and precision of both methods throughout the working range are presented. BAY x 7195 is stable in plasma after repeated freeze-thaw cycles and upon storage at −20°C for at least 13 months. Method A was applied to a clinical study with oral administration of 250 mg BAY x 7195 where ca. 1% of the maximum plasma concentrations still could be accurately and precisely quantified. Method B was employed to determine the drug in plasma after administration of 1 mg as aerosol.     

Rapid and sensitive determination of amprolium in chicken plasma by high-performance liquid chromatography with post-column reaction

A rapid, sensitive and reproducible reversed-phase HPLC assay was developed for the determination of amprolium (APL) in chicken plasma. Protein in plasma sample was precipitated with 0.33 M perchloric acid and supernatant solution was injected into the HPLC system. Following the chromatographic separation of APL and the beclotiamine (I.S.) on a C₁₈ column, the derivatives of APL and I.S. were formed by post-column reaction and detected by fluorescence detection (excitation at 400 nm, emission at 460 nm). The method showed excellent precision, accuracy and speed with a detection limit of 2 ng/ml. The intra- and inter-assay variance of this method were less than 11.2%. This method has been successfully applied to plasma determinations after oral administration of APL to chicken.     

Improved method for the rapid determination of isosorbide dinitrate in human plasma and its application in pharmacokinetic studies

A rapid, accurate and highly sensitive method was developed for the determination of isosorbide dinitrate in human plasma. Concentrations in the lower nanogram and subnanogram range are determined by a one-step extraction of 2 ml plasma, containing 4 ng/ml nitroglycerine as internal standard, with 5.5 ml n-pentane. The extract is subjected to gas—liquid chromatography—electron capture detection analysis. The lower limit of quantitation is 200 pg/ml, but concentrations as low as 50 pg/ml are still detectable. The method allows the quantitative determination of isosorbide dinitrate plasma levels in man following a 5 mg sublingual administration up to four hours after application.     

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step – ultrafiltration for plasma and dilution for urine samples – followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

High-performance liquid chromatographic determination of ambroxol in human plasma

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid—liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

On-line dialysis and weak cation-exchange enrichment of dialysate : Automated high-performance liquid chromatography of pholcodine in human plasma and whole blood

An automated method for the determination of pholcodine in plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to high-performance liquid chromatography. Dialysis, trace enrichment on a weak cation-exchange column, separation on a cyano column and fluorescence detection was shown to be an extremely selective and sensitive method. The method has been used successfully in the analysis of real samples after administration of pholcodine. The automated method can be used, after minor modification, to determine other basic drugs in whole blood and plasma.     

Quantitative determination of imidazenil,a novel imidazobenzodiazepine car☐amide derivative,by normal-phase high-performance liquid chromatography

Imidazenil, an imidazobenzodiazepine car☐amide derivative, is a novel anxiolytic and anti-convulsant agent recently characterized as a partial allosteric modulator of GABA_A receptors. Owing to the pharmacological and pharmacokinetic importance of plasma-level determination, a HPLC method has been developed. Imidazenil was extracted from a plasma sample after a partition with diethyl ether, using alprazolam as internal standard. The analysis was performed by a normal-phase HPLC method with UV detection at 255 nm. The limit of quantitation was 6 ng corresponding to 30 ng/ml of plasma concentration. This procedure has been successfully applied to the quantitation of imidazenil plasma levels in primarily pharmacokinetic studies after a single i.v. and an oral administration of the compound to the rat.     

Determination of 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)-dodecanamide, a lipid regulator, in rat plasma and mesenteric lymph by reversed-phase high-performance liquid chromatography

2,2-Dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (I, CI-976) has been determined in rat mesenteric lymph and plasma using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma and lymph by liquid—liquid extraction were analysed on a reversed-phase C₁₈ column using isocratic conditions and ultraviolet detection. The method was applied to the determination of levels of I in Wistar rats after intraduodenal administration of 110 mg/kg of I as a lipid emulsion.     

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.     

Gas chromatographic determination of the hypoglycaemic agent gliclazide in plasma

A gas chromatographic method has been developed that permits the accurate and specific determination of the hypoglycaemic agent gliclazide in plasma. Gliclazide is extracted with chloroform and, after clean-up, derivatized with diazomethane followed by heptafluorobutyric anhydride to form N-methyl-N′-heptafluorobutyrylgliclazide, which is assayed on a gas chromatograph equipped with a flame ionization detector, an electron-capture detector or a nitrogen—phosphorus sensitive detector.Accurate determinations are possible with flame ionization detection over a concentration range of 1–15 μg/ml of gliclazide in plasma with a relative standard deviation of 5.2%. The minimum detectable concentration with electron-capture detection is 0.02 μg per sample. Plasma levels of gliclazide in dogs following single oral administration (40 mg per dog) have also been determined.     

Simultaneous Determination of Dermatan Sulfate and Oversulfated Dermatan Sulfate in Plasma by High-Performance Liquid Chromatography with Postcolumn Fluorescence Derivatization

A chemical method for the determination of dermatan sulfate (DS) and oversulfated dermatan sulfate has been developed and applied to the pharmacokinetic study of these polysaccharides in experimental animals. The analytical procedure includes a simple preparation step of administered DS and oversulfated DS from blood plasma, HPLC for the separation and detection of DS and oversulfated DS using an Asahipak NH₂P-50 column, fluorometric reaction of the polysaccharides with guanidine in a strong alkaline medium. DS and oversulfated DS were extracted from plasma by treating it with proteinase to remove plasma proteins and recovered with endogenous plasma glycosaminoglycans by ethanol precipitation. Finally, DS and oversulfated DS were analyzed by fluorometric HPLC. The detection limits of DS and oversulfated DS were 10 and 20 ng, respectively. Furthermore, we demonstrated that artificial oversulfation of DS increased its biological half-life after intravenous administration to rats.     

Pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration

Amlodipine, 3-ethyl 5-methyl-2-[(2-aminoethoxymethyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, is a chiral calcium antagonist, currently on the market and in therapeutic use as a racemate. The pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration to healthy male human volunteers together with comparative administration of the racemic mixture of both enantiomers were studied. Plasma levels were studied as a function of time and assayed using an enantioselective chromatographic method (coupled chiral and achiral HPLC) with on-line solid-phase extraction and UV absorbance detection. The method was validated separately for the R-(+)- and S-(−)-enantiomer, respectively. Results of the study indicate that the pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration is comparable to that of each enantiomer after administration of the racemate. No racemization occurs in vivo in human plasma after single enantiomer administration.     

High-performance liquid chromatographic assay for the determination of 5-methyltetrahydrofolate in human plasma

An isocratic high-performance liquid chromatographic method for the determination of 5-methyltetrahydrofolate (5-MTHF) in human plasma is described. The method involves solid-phase extraction of 5-MTHF and p-aminoacetophenon (an internal standard) using Sep-Pak C₁₈ cartridges. Separation was achieved with an ODS column using acetonitrile and phosphate buffer supplemented with octanesulfonic acid (an ion-pairing agent). The pH of the mobile phase (2.5) was optimal with respect to the mode of detection (fluorescence). The method was validated in the range of 5-MTHF concentrations from 0.0625 μmol/l to 4.0 μmol/l. Within-day and inter-day precision expressed by the relative standard deviation was less than 8.1% and inaccuracy did not exceed 8.7%. The method is specific, accurate and sensitive enough to be used in pharmacokinetic studies for the assessment of the systemic availability of 5-MTHF after leucovorin administration to patients as a rescue after high-dose therapy with methotrexate. The limit of detection was 0.17 pmol which corresponds to a plasma concentration of 1.7 nmol/l. Thus, the assay could potentially be used for the measurement of 5-MTHF in the range of physiological concentrations in plasma (5–20 nmol/l).     

Determination of the α,β-adrenoceptor blocker YM-09538 in plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of the α,β-adrenoceptor blocker 5-{1-hydroxy-2-[2-(o-methoxyphenoxy)ethylamino]ethyl}-2-methylbenzenesulphonamide hydrochloride (YM-09538) in plasma, using 5-di-n-butylaminonaphthalene-1-sulphonyl chloride as a reagent for fluorescence labelling, is described. The detection limit is 20 ng/ml, which is sensitive enough to determine YM-09538 plasma levels after the oral administration of effective doses to dogs and humans.     

Determination of Z-butylidenephthalide in plasma by high-performance liquid chromatography

An HPLC assay is described for the determination of Z-butylidenephthalide (Z-Bdph) in plasma. Plasma samples were cleaned up by extraction with 2% chloroform in n-hexane. Z-Bdph was separated on a normal-phase silica column with a mobile phase of chloroform-n-hexane (1:1). The limit of quantitation with UV detection at 254 nm for Z-Bdph in plasma was 0.01 μg/ml. The recovery of Z-Bdph by organic solvent extraction of plasma was 99.5%. The intra-day and inter-day coefficients of variation and relative errors for Z-Bdph determination in plasma were both less than 10%. The present method was applied to pharmacokinetic studies of Z-Bdph in plasma after intravenous administration to rabbits.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.